Branched pei

HEK293T cells were seeded in 96 well plates and transfected with either a plasmid encoding the full-length SARS-CoV-2 spike (pLVX-EF1alpha-SARS-CoV-2-S-2xStrep-IRES-Puro) or mock plasmid (pcDNA3.1) using branched PEI (Sigma). Media was switched 24 h post-transfection. At 48 h post-transfection, cells were washed 5 times with PBS prior to fixation with 4% paraformaldehyde in media for 30 min at 4 °C. All further washing steps were performed 5 times with PBS + 0.05% Tween 20 (PBS-T). Cells were washed prior to blocking in blocking buffer (PBS-T + 2% BSA) for 1 h at room temperature. After washing, cells were incubated with dilutions of primary antibodies in blocking buffer for 2 h at room temperature. After washing, cells were incubated with goat anti-human IgG (Jackson ImmunoResearch) at a 1:5,000 dilution in blocking buffer for 1 h at room temperature. After washing, substrate solution (Pierce 1-Step) was used for colour development according to the manufacturer's specifications. Optical density at 450 nm was read on a Varioskan Lux plate reader (Thermo Fisher Scientific). The difference in signals between cells transfected with full-length spike and mock plasmid was calculated.

Branched PEI with molecular weights of 25 and 750 kDa as well as 2,4,6-trinitrobenzenesulfonic acid (TNBSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (D-MEM), trypsin-EDTA, fetal bovine serum (FBS), phosphate-buffered saline (PBS) were obtained from Gibco (Carlsbad, CA, USA). Pyrrole and ammonium persulfate (APS) were purchased from Acros (Geel, Belgium) and Showa (Tokyo, Japan), respectively.